Collins, C.R.; (2005) Functional studies of the plasmodium falciparum apical membrane antigen 1. Doctoral thesis , University of London.

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Abstract

Host cell invasion by apicomplexan parasites involves multiple molecular interactions at the parasite-host cell interface, many of which provide likely targets for drug or vaccine-mediated intervention. Apical membrane antigen-1 (AMA-1) is an essential, widely conserved type I integral membrane protein which is mobilised onto the parasite surface from microhenries just prior to invasion. Antibodies to AMA-1 can prevent invasion. In the case of the malaria parasite, antibodies to AMA-1 can also protect against blood-stage infection in vivo and Plasmodium falciparum AMA-1 (PfAMA-1) is a prime candidate for inclusion in a blood-stage malaria vaccine. Rational vaccine design would benefit from a clearer understanding of PfAMA-1 function and the delineation of functionally important domains of the protein. A COS-7 cell- surface expression system, combined with a mutagenesis-based screen, was used to identify a series of residues critical for the binding of a monoclonal antibody that interferes with PfAMA-1 function. Most of these residues lie in a single disulfide-constrained domain of the molecule. Our data indicate that this domain is of particular functional and immunological significance. In contrast to studies of AMA1 from other species of Plasmodium, we have been unable to identify a role for PfAMAI in erythrocyte binding using this system. This result may be due to the absence of essential parasite factors in this heterologous system. Furthermore, it has not been possible to identify any associated parasite proteins. Thus, the role of AMA1 in invasion is still unclear. Much of this work has relied on use of the Pfamal synthetic gene (sgPfamal). To validate the use of this gene sequence and show functionality of the gene product in the parasite, transfection studies were carried out. Attempts are underway to replace the endogenous gene sequence with a haemagglutinin (HA) tagged version of the synthetic gene. Expression of the sgPfamal gene product has been confirmed by immunofluorescence assay, as well as correct localisation to the apical end of the parasite. Disruption of the endogenous gene sequence remains to be confirmed.

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